Bacteria in many bacterial genera, for example Erwinia, Pseudomonas, Xanthomonas, Agrobacterium, Azotobacter, Flavobacterium and Bacillus, are known to interact with plants. Many types of bacteria and other microbes also infect plants, resulting in plant diseases. Microbial plant diseases may be suppressed by so-called "bio-control" bacteria. Bio-control bacteria either grow more competitively in the plant environment or on the plant surface than the pathogenic microbes, or may produce inhibitory substances that decrease the virulence of the pathogenic microbes or inhibit their growth. A knowledge of the genes of the pathogens or of the bio-control bacteria that cause them to multiply in response to a root or other plant part may allow better understanding of the cause of pathogenesis as well as allowing better understanding and the selection of bio-control bacteria better able to compete against the pathogens in the natural environment.
Use of "marker" genes has enabled researchers to determine the fate of marked microorganisms when added to a soil population of the same microorganisms not having the marker gene. These marker genes may be caused by mutagenesis of the wild-type cells by chemical treatment, genetic transformation (U.S. Pat. No. 4,753,876), or other means. This patent and all other patents, papers and books cited herein are hereby incorporated herein by reference.
It is known that certain genes of bacteria may be induced by the host plants. Osbourn et al. (EMBO J. 6:23-28, 1987) constructed a broad host range promoter-probe plasmid containing a promoterless chloramphenicol acetyltransferase (CAT) gene and introduced this gene on the plasmid into Xanthomonas to identify two classes of Xanthomonas promoters. Seedlings treated with chloramphenicol and inoculated with Xanthomonas were used to select for chloramphenicol resistance of the bacteria expressed in planta, with only the resistant Xanthomonas bacteria (having an active promoter upstream of the CAT gene) being able to cause symptoms on the plants in the presence of chloramphenicol. This test requires a plant assay and analysis of plant damage to screen mutants for those with active promoters upstream of the CAT gene. Plasmids also tend to be unstable in a host bacterium and may be lost.
Schilperoort et al. (European Patent Application Number 0 167 192 A1, published Jan. 8, 1986) utilized a promoter region of a virulence gene of Agrobacterium tumefaciens linked with a promoterless lacZ gene to explore induction of the selected bacterial promoter. The resultant plasmid was placed in E. coli and Agrobacterium and the cells were exposed to plant substances. It was found that plant substances caused the inserted promoter to be induced resulting in expression of beta-galactosidase activity of the lacZ gene. This procedure allows testing of a selected promoter but does not enable identification of unknown promoters that may be induced by plant substances.
Transposons have been used to study the genetics of bacteria, including their ability to cause plant nodulation or their competitive ability. Meade et al. (J. Bacteriol. 149:114-122, 1982) utilized transposons in Rhizobium meliloti and characterized the symbiotic and auxotrophic nature of the resulting mutants. Innes et al. (Molecular Gen. Genetics 201:426-432, 1985) isolated lac operon transcriptional fusions to a number of genes within a fragment of the R. trifolii symbiosis plasmid using a bacteriophage transposon, and monitored expression of the lac gene in the various positions in this fragment in response to the plant.
In co-pending application, Ser. No. 07/244,813, filed Sep. 14, 1988, Pseudomonas bacteria were mutagenized with a transposon (Tn5-derivative) containing a marker gene, so that mutants having the transposon constitutively expressed the marker gene. Mutant strains were screened for competitiveness on the plant using plant inoculation studies with mixtures of wild-type and mutant cultures. The transposon-linked genes of mutants having alterations in their competitiveness as compared to the wild-type strain were cloned.
As used in the co-pending application and herein, transposons, or transposable elements, are DNA segments that can move (transpose) in the genome and insert into different sites on the chromosome without benefit of homology. They can also be moved from one bacterium to another bacterium by plasmid vehicles to be inserted into the host genome.
Simple transposons, composed of insertion sequences (IS) contain no genes other than those involved in their own transposition into the target genome. More complex transposons contain IS elements bracketing additional genes that encode properties such as drug resistance, carbohydrate metabolism, light generation, ice nucleation, or other properties, which can function as selectable or screenable markers for the entire transposable element. If two IS elements bracket a segment of DNA, the entire segment, including both flanking IS elements, may be able to transpose as a unit and the entire segment is then considered a transposon.
Transposons appear to be present in the genome of all types of organisms and occurs naturally in a wide variety of types. Many naturally occurring transposons are of a very complex structure. They are generally integrated into the genome of the cell, and can transpose in the genome and insert at different sites. This insertion may be at a particular DNA site or may be nonspecific or general insertion. When the insertion of the transposable element into a particular gene affects the expression of the gene, a mutation occurs.
Transposons which insert nonspecifically are useful for causing generalized mutagenesis. The transposon, Tn5, containing a gene coding for kanamycin resistance, was demonstrated by Beringer et al., Nature, 276:633-634, 1978) to be a suitable transposon for generalized transposon-insertion mutagenesis in Rhizobium leguminosarum, being carried into these nodule-causing bacterial by a plasmid able to infect gram negative bacteria.
It is therefore an object of this invention to provide a method whereby bacterial genes responsive to a plant host may be identified utilizing a plant-derived substance.
It is a further object of this invention to provide a method utilizing root exudates to identify plant-responsive bacterial genes in the absence of the plant.
It is a further object of this invention to provide bacterial genes responsive to plant hosts.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.